Vector Selector

Different combinations of expression vector elements yield different protein expression levels. Vector behavior also depends on the expression cell line used. We have tested hundreds of combinations for their performance in HEK and CHO cells. You can select the best of these vectors from the interactive graph below. For other cell lines, we recommend a vector panel to determine the best performing vector for your system.

Antibody Expression from a Single Vector

Transient Vectors Show Range of Expression in HEK293 and CHO Cells

Antibody production in transiently transfected HEK293 and CHO (suspension) cells. ATUM dual expression vector configurations (single construct) enable co-expression of antibody’s heavy and light chains by coupling expression through an IRES. Cells were transfected at 2 x 106 cells/ml using Fugene; transfections were carried out in triplicate in HEK293 and CHO (suspension) cells. Antibody expression was measured by heavy chain quantitation by ELISA, 6 days post-transfection.

Comparison of Antibody Expression from Single Constructs

The balance of expression of an antibody’s heavy and light chains has a profound effect on antibody yield. The two chains can be co-expressed by coupling expression through an IRES, or by using a dual promoter configuration. Co-transfection of separate vectors carrying heavy and light chains is also feasible for transiently transfected cells, but is less desirable for stable cell line generation. Integrated copy number and integration position effects affect the balance of expression levels of the two chains, compromising the yield of properly assembled antibody. ATUM dual expression vector configurations outperform co-transfections of separate heavy and light chains.

Antibody production from transiently transfected HEK293 cells.IRES element in pD2610-v6 (pink bar) or dual promoter configuration (blue bar) were used to drive expression of both antibody chains from the same construct. Co-transfections of 3 ratios of heavy:light chain constructs (grey bars) are shown. Antibody was measured by ELISA from the media 6 days post-transfection.

Bicistronic Expression

Two or more proteins can be expressed from a single promoter using either IRES or 2A/CHYSEL translational-coupling sequences. Constructs using translational coupling elements are often smaller and less repetitive than those which use a separate promoter for each protein. IRESes and CHYSEL elements have different properties that make them suitable for different applications.

IRES Elements

IRES (Internal Ribosome Entry Site) elements create a second, cap-independent site for ribosomes to bind an mRNA molecule. This results in independent expression of two proteins from a single message. The ratio at which the two proteins are expressed depends on the efficiency of ribosome entry at the IRES compared with cap-dependent scanning from the 5′ end of the mRNA. Different IRESes provide different expression ratios of the 2 proteins (beige and blue arrows) and can be chosen based upon the ratio that is desired.

IRESes are particularly useful for antibody expression, where the optimal ratio between the chains for efficient assembly can differ for different antibodies. ATUM has developed a set of novel IRES elements, each produces a defined expression ratio between the first and second protein in every transfected cell. We can help you choose from these IRES elements to design and synthesize a custom bicistronic expression system with controlled protein expression ratios.

IRESes can also link expression of a protein to a selectable marker or fluorescent reporter expressed from a stably integrated vector, to indicate whether the construct is integrated at high copy numbers and in positions that are good for expression.

DasherGFP and CayenneRFP expression compared in transiently transfected CHO and HEK293 cells. IRES elements were tested in a GFP-IRES-RFP configuration in CHO (blue circles) and HEK293 (green circles) cells. Cells were harvested for DasherGFP and CayenneRFP fluorescence measurements 72 hours post-transfection. The expression ratio was normalized using the ratio for sequences separated by a CHYSEL site. IRES efficiency is the ratio of RFP:GFP. An IRES can be selected to control the ratio of bicistronic expression between two proteins.

CHYSEL (2A) Elements

CHYSEL 2A elements cause ribosomes to release the first protein while remaining bound to the mRNA and continuing translation. They therefore produce approximately equimolar amounts of the two (or more) proteins. Because ribosome binding occurs only at the start of the entire construct and reads through both ORFs, these are particularly useful for monitoring expression levels of a protein. Some ATUM vectors have a cloning site upstream or downstream of a gene encoding a fluorescent protein (green arrow), whose expression is directly proportional to the level of the other protein (beige arrow). CHYSEL elements add 18 amino acids to the C-terminus of the first protein, which can compromise the activity of some proteins. When used for antibody expression this can sometimes lead to formation of insoluble aggregates.

pD2600 Overview

ATUM has developed a new family of transient expression vectors by using VectorGPS™ to test combinations of vector elements for their performance in HEK and CHO cells. Many of these combinations yield higher expression than pCDNA3.4 or our first generation pJ600 series. The way to access these vectors:

Get your gene synthesized and cloned into a vector. pD2600 vectors work reliably in both HEK and CHO cells.

Test several vectors to see which works best in your cell line. Vectors with DasherGFP optimized for mammalian expression are also available to test expression.

Perform VectorGPS™ to design and build the optimal vector for your protein and cell line.

Vector Cost

Positive control vectors are available from ATUM’s catalog at half price if ordered with gene synthesis orders.

pD600s

ATUM′s pD600 series of mammalian transient expression vectors are still available. These vectors use the strong CMV/IE enhancer/promoter and an SV40 replication origin to give high levels of protein expression in mammalian cells. Although most pD600s have mammalian selectable markers, we recommend the Bifunctional Leap-In™ vectors for generating stable cell lines. pD600 vectors are available with DasherGFP® as a positive control. Cloning into these vectors is performed at no additional cost for gene synthesis orders.

Notes

Important Info: Vectors used for Gene Synthesis orders do NOT contain the multiple cloning sites.

Restriction Sites: The pJ and pD vectors do not contain restriction sites for excision of your gene. Any restriction site desired can be added to your synthetic gene insert, and must be included in your gene design if you wish to remove your gene with restriction enzymes.

pD2600 Products (Research Use Only Products):
License for ATUM Research Use Only Vectors. Except as set forth and subject to the terms herein, if Customer purchases (1) any ATUM Product containing pD2100, pD2500, pD2600, 3400, pD3500, 3600 or 3700 series vectors, or a vector containing the FH8 solubility tag (the “ATUM Research Use Only Vectors”) (including Electra vectors, vector configurations for expression of multiple genes and other customized configurations of the ATUM Research Use Only Vectors, and Customer genes cloned into the ATUM Research Use Only Vectors) or (2) any ATUM Service that involves/utilizes ATUM Research Use Only Vectors, Customer is granted a limited, non-sublicensable, non-transferable, non-exclusive right to use the purchased amount of the ATUM Research Use Only Vectors to produce proteins for research and development purposes only. Customer shall not and shall not cause or permit any third party to (1) modify ATUM Research Use Only Vectors or any product containing ATUM Research Use Only Vectors in any way, including without limitation by replacing any protein-encoding sequence with any other protein-encoding sequence within the ATUM Research Use Only Vector and/or replacing the gene cloned into the ATUM Research Use Only Vectors; (2) directly or indirectly reverse-engineer the ATUM Research Use Only Vectors or any product containing the ATUM Research Use Only Vectors; (3) create any derivative(s) of the ATUM Research Use Only Vectors; (4) copy the ATUM Research Use Only Vectors or (5) use the ATUM Research Only Vectors for commercial purposes. Use of the ATUM Research Only Vectors for commercial purposes, including any use in manufacturing a product or service that is provided to a third party for consideration, requires a commercial license from ATUM.

Vector Selector

Different combinations of expression vector elements yield different protein expression levels. Vector behavior also depends on the expression cell line used. We have tested hundreds of combinations for their performance in HEK and CHO cells. You can select the best of these vectors from the interactive graph below. For other cell lines, we recommend a vector panel to determine the best performing vector for your system.

Overview

The organization and orientation of functional elements in our new stable expression vectors promote efficient stable cell-line generation and high expression levels both before and after integration. These vectors are available with a choice of highly active promoters and mammalian selection markers.

Expression of 2, 3 or 4 ORFs from a Single Vector

Two or more proteins can be expressed simultaneously from a single vector using two or more promoters. We can help you choose from several available promoter elements to design and synthesize a custom expression system with controlled protein expression ratios. For example, modulate antibody heavy and light chain expression levels in a single construct or to express all of the chains for bispecifics. Contact ATUM Ph.D. level customer support to assist with design.

Two identical ORFs coding for a secreted protein were tagged with V5 (ORF1) or FLAG (ORF2). A combination of vector elements were utilized to control the expression ratio of ORF2 to ORF1. Expression ratios of ORF2:ORF1 between 2.5:1 and 1:1.5 were demonstrated.

ORFs encoding 3 fluorescent proteins were cloned into six different 3-ORF vectors. Fluorescence was measured at the stable pool stage. Various ratios of all three ORFs were achieved.

ORFs encoding 4 fluorescent proteins were cloned into a single 4-ORF vector. Fluorescence was measured at the stable pool stage. A 4-fold range of expression was observed between the different ORFs.

Legend

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DasherGFP expression in stably transfected CHO cells and transient expression in HEK293 cells. Cells were transfected at 70-80% confluency using Lipofectamine 2000; transfections were carried out in triplicate in HEK293 cells and CHO cells. Selection media with 5 µg/ml Puromycin was added 72 hours post-transfection, grown for an additional 72 hours post-selection and media changed to complete growth media for a total of 14 days (2 passages in complete growth media) for CHO cells. DasherGFP transient expression was measured 72 hours post-transfection in HEK293 cells. Cells were lysed using M-PER and expression measured on a fluorimeter.

HPRE Outperforms WPRE

DasherGFP Expression in Stably Transfected CHO Cells

DasherGFP expression in vectors with either the HPRE or WPRE enhancer elements in stably transfected CHO cells. Cells were transfected at 70-80% confluency using Lipofectamine 2000; transfections were carried out in triplicate. Selection media with 5 µg/ml Puromycin was added 72 hours post-transfection, grown for an additional 72 hours post-selection and media changed to complete growth media for a total of 14 days (2 passages in complete growth media). Cells were lysed using M-PER and expression measured on a fluorimeter.

Vector Cost

Positive control vectors expressing DasherGFP are available from ATUM’s catalog at half price if ordered with gene synthesis orders.

Notes

Important Info: Vectors used for Gene Synthesis orders do NOT contain the multiple cloning sites.

Restriction Sites: The pD vectors do not contain restriction sites for excision of your gene. Any restriction site desired can be added to your synthetic gene insert, and must be included in your gene design if you wish to remove your gene with restriction enzymes.

License for ATUM Research Use Only Vectors:
Except as set forth and subject to the terms herein, if Customer purchases (1) any ATUM Product containing pD2100, pD2500, pD2600, 3400, pD3500, 3600 or 3700 series vectors, or a vector containing the FH8 solubility tag (the “ATUM Research Use Only Vectors”) (including Electra vectors, vector configurations for expression of multiple genes and other customized configurations of the ATUM Research Use Only Vectors, and Customer genes cloned into the ATUM Research Use Only Vectors) or (2) any ATUM Service that involves/utilizes ATUM Research Use Only Vectors, Customer is granted a limited, non-sublicensable, non-transferable, non-exclusive right to use the purchased amount of the ATUM Research Use Only Vectors to produce proteins for research and development purposes only. Customer shall not and shall not cause or permit any third party to (1) modify ATUM Research Use Only Vectors or any product containing ATUM Research Use Only Vectors in any way, including without limitation by replacing any protein-encoding sequence with any other protein-encoding sequence within the ATUM Research Use Only Vector and/or replacing the gene cloned into the ATUM Research Use Only Vectors; (2) directly or indirectly reverse-engineer the ATUM Research Use Only Vectors or any product containing the ATUM Research Use Only Vectors; (3) create any derivative(s) of the ATUM Research Use Only Vectors; (4) copy the ATUM Research Use Only Vectors or (5) use the ATUM Research Only Vectors for commercial purposes. Use of the ATUM Research Only Vectors for commercial purposes, including any use in manufacturing a product or service that is provided to a third party for consideration, requires a commercial license from ATUM.

Vector Selector

Overview

ATUM’s new series of lentiviral expression vectors (pD2100s) deliver efficient integration and high expression with HPRE and AGS elements.
Lentiviral expression vectors integrate a defined region of plasmid vector into a target cell line. They are available with DasherGFP as a positive control. Lentiviral vectors infect both dividing and non-dividing cells, producing homogenous levels of transgene expression across a population of cells. Available for gene synthesis orders, and as ready-to-use catalog vectors.

Lentiviral Packaging

ATUM does not supply packaging plasmids or cell lines, however ATUM Lenti vectors are compatible with both 2nd and 3rd generation lentiviral packaging systems. Minimally, plasmids encoding HIV-1 Gag, Pol, Rev and envelope (VSV-G is recommended) are necessary for co-transfection with Lenti vector for proper formation of infectious viral particles. HEK293 or a derivative (293T, etc.) cell line are recommended to allow for high efficiency simultaneous transfection of 3-4 plasmids.

See datasheet for an example of a protocol that has been effective for 2nd generation packaging systems.

Notes

Important Info: Vectors used for Gene Synthesis orders do NOT contain the multiple cloning sites.

Restriction Sites: The pD vectors do not contain restriction sites for excision of your gene. Any restriction site desired can be added to your synthetic gene insert, and must be included in your gene design if you wish to remove your gene with restriction enzymes.

pD2100 Products (Research Use Only Products):
License for ATUM Research Use Only Vectors. Except as set forth and subject to the terms herein, if Customer purchases (1) any ATUM Product containing pD2100, pD2500, pD2600, 3400, pD3500, 3600 or 3700 series vectors, or a vector containing the FH8 solubility tag (the “ATUM Research Use Only Vectors”) (including Electra vectors, vector configurations for expression of multiple genes and other customized configurations of the ATUM Research Use Only Vectors, and Customer genes cloned into the ATUM Research Use Only Vectors) or (2) any ATUM Service that involves/utilizes ATUM Research Use Only Vectors, Customer is granted a limited, non-sublicensable, non-transferable, non-exclusive right to use the purchased amount of the ATUM Research Use Only Vectors to produce proteins for research and development purposes only. Customer shall not and shall not cause or permit any third party to (1) modify ATUM Research Use Only Vectors or any product containing ATUM Research Use Only Vectors in any way, including without limitation by replacing any protein-encoding sequence with any other protein-encoding sequence within the ATUM Research Use Only Vector and/or replacing the gene cloned into the ATUM Research Use Only Vectors; (2) directly or indirectly reverse-engineer the ATUM Research Use Only Vectors or any product containing the ATUM Research Use Only Vectors; (3) create any derivative(s) of the ATUM Research Use Only Vectors; (4) copy the ATUM Research Use Only Vectors or (5) use the ATUM Research Only Vectors for commercial purposes. Use of the ATUM Research Only Vectors for commercial purposes, including any use in manufacturing a product or service that is provided to a third party for consideration, requires a commercial license from ATUM.